A number of serious diseases, including infectious diseases, and even certain types of cancer, utilize proteolytic enzymes in physiological functions that play a critical role in their life cycles. Aspartic proteases are among the proteolytic enzymes that have been identified in this connection. In order to combat diseases which utilize aspartic proteases in critical aspects of their life cycles, aggressive efforts have been undertaken to develop aspartic protease inhibitors, particularly over the last decade. Recent efforts in this area have primarily focused in the treatment or prevention of acquired immune deficiency syndrome (AIDS). AIDS is a fatal disease, reported cases of which have increased dramatically within the past several years. Estimates of reported cases in the very near future also continue to rise dramatically.
The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphocyte virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. This particular retrovirus is also known as lymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2. The acronym HIV will be used herein to refer to HIV viruses generically.
Specifically, HIV is known to exert a profound cytopathic effect on the CD4+ helper/inducer T-cell—thereby severely compromising the immune system. HIV infection also results in neurological deterioration and, ultimately, in the death of the injected individual
The field of viral chemotherapeutics has developed in response to the need for agents effective against retroviruses, in particular HIV. For example anti-retroviral agents, such as 3′-azido-2′,3′-dideoxythymidine (AZT), 2′3′-dideoxycytidine (ddC), and 2′3′-dideoxyinosine (ddI) are known to inhibit reverse transcriptase. There also exist antiviral agents that inhibit transactivator protein. Nucleoside derivatives, such as AZT, are currently available for antiviral therapy. Although very useful, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy.
Retroviral protease inhibitors also have been identified as a class of anti-retroviral agents. Retroviral protease is an aspartic protease that processes polyprotein precursors into viral structural proteins and replicative enzymes. This processing is essential for the assembly and maturation of fully infectious virions. Accordingly, the design of such aspartic protease inhibitors remains an important therapeutic goal in the treatment of AIDS.
The use of HIV protease inhibitors, in combination with agents that have different antiretroviral mechanisms (e.g., AZT, ddI and ddT), also has been described. For example, synergism against HIV-1 has been observed between certain C2 symmetric HIV inhibitors and AZT (Kageyama et al., Antimicrob. Agents Chemother., 36, 926–933 (1992)).
Numerous classes of potent peptidic inhibitors of protease have been designed using the natural cleavage site of the precursor polyproteins as a starting point. These inhibitors typically are peptide substrate analogs in which the scissile P1–P1′ amide bond has been replaced by a non-hydrolyzable isostere with tetrahedral geometry (Moore et al, Perspect. Drug Dis. Design, 1, 85 (1993); Tomasselli et al., Int. J. Chem. Biotechnology, 6 (1991); Huff, J. Med. Chem., 34, 2305 (1991); Norbeck et al., Ann. Reports Med. Chem., 26, 141 (1991); and Meek, J. Enzyme Inhibition, 6, 65 (1992)). Although these inhibitors are effective in preventing the retroviral protease from functioning, the inhibitors suffer from some distinct disadvantages. Generally, peptidomimetics often make poor drugs, due to their potential adverse pharmacological properties, i.e., poor oral absorption, poor stability and rapid metabolism (Plattner et al, Drug Discovery Technologies, Clark et al., eds., Ellish Horwood, Chichester, England (1990)).
The design of the HIV-1 protease inhibitors based on the transition state mimetic concept has led to the generation of a variety of peptide, derivatives highly active against viral replication in vitro (Erickson et al, Science, 249, 527–533 (1990); Kramer et al., Science, 231, 1580–1584 (1986); McQuade et al., Science, 247, 454–456 (1990); Meek et al., Nature (London), 343, 90–92 (1990); and Roberts et al., Science, 248, 358–361 (1990)). These active agents contain a non-hydrolyzable, dipeptidic isostere, such as hydroxyethylene (McQuade et al., supra; Meek et al., Nature (London), 343, 90–92 (1990); and Vacca et al., J. Med. Chem., 34, 1225–1228 (1991)) or hydroxyethylamine (Ghosh et al., Bioorg. Med. Chem. Lett., 8, 687–690 (1998); Ghosh et al., J. Med. Chem., 36, 292–295 (1993)); Rich et al., J. Med. Chem., 33, 1285–1288 (1990); and Roberts et al., Science, 248, 358–361 (1990)) as an active moiety that mimics the putative transition state of the aspartic protease-catalyzed reaction.
Two-fold (C2) symmetric inhibitors of HIV protease represent another class of potent HIV protease inhibitors on the basis of the three-dimensional symmetry of the enzyme active site (Erickson et al. (1990), supra). Typically, however, the usefulness of currently available HIV protease inhibitors in the treatment of AIDS has been limited by relatively short plasma half-life, poor oral bioavailability, and the technical difficulty of scale-up synthesis (Meek et al. (1992), supra).
In a continuing effort to address the problem of short plasma half-life and poor bioavailability, new HIV protease inhibitors have been identified. For example, HIV protease inhibitors incorporating the 2,5-diamino-3,4-disubstituted-1,6-diphenylhexane isostere are described in U.S. Pat. No. 5,728,718 (Randad et al.). HIV protease inhibitors, which incorporate the hydroxyethylamine isostere, are described in U.S. Pat. No. 5,502,060 (Thompson et al.), U.S. Pat. No. 5,703,076 (Talley et al.), and U.S. Pat. No. 5,475,027 (Talley et al.).
The emergence of mutant strains of HIV, in which the protease is resistant to the C2 symmetric inhibitors, also presents a serious challenge in the treatment and prevention of AIDS (Otto et al., PNAS USA, 90, 7543 (1993); Ho et al., J. Virology, 68, 2016–2020 (1994); and Kaplan et al., PNAS USA, 91, 5597–5601 (1994)). In one study, the most abundant mutation found in response to a C2 symmetry based inhibitor was Arg to Gln at position 8 (R8Q), which strongly affects the S3/S3′ subsite of the protease binding domain.
Other mutant viruses have been identified, for example, in which the amino acid at position 84 of the retroviral protease has mutated from isoleucine into valine (84V), and in which the amino acid at position 32 of the retroviral protease has mutated to include valine (84V). Double mutants, for example, 82F/84V also have been identified. Thus, it is desirable to identify new aspartic protease inhibitors in the continuing effort to combat AIDS, and to address the problems emerging with the rise of mutant HIV strains.
Aspartic proteases also have been implicated in the life cycle of other serious infectious diseases, for example, Malaria. Malaria is one of the worlds most devastating diseases, afflicting several hundred million people a year, and killing an estimated two million of those infected, mostly children. The etiologic agent is a parasitic protozoan of the genus Plasmodium, which causes disease in its intraerythrocytic phase. A prominent feature of its development inside red blood cells is the degradation of hemoglobin. During its relatively short life cycle, the malaria-causing parasite consumes nearly all of the host's hemoglobin, generating amino acids for its own growth and maturation. The degradation of the host's hemoglobin, which is a vast catabolic process, involves a series of proteases, two of which have been identified as aspartic proteases termed “plasmepsins.” The structure of plasmepsin II has recently been elucidated on the basis of diffraction data Silva et al., Aspartic Proteinases, 51, pp. 363–373, New York (1998).
Although potent plasmepsin inhibitors have been identified, their ability to inhibit the malarial parasite in culture is very limited, possibly due to the inability of the inhibitors to penetrate the cellular structure sufficiently to inhibit plasmepsin in the microorganism itself. See, e.g., Silva et al., Proc. Nat. Acad. Sci. USA, 93, pp. 10034–10039, September 1996 (Biochemstry). Metabolism of the inhibitor by the microorganism also may be a factor. Thus, it remains a challenge to identify new plasmepsin inhibitors that are potent and also are effective against the malarial parasite in culture.
Aspartic proteases also have been implicated in essential pathways related to cancer. For example, elevated levels of cathepsin D, an aspartic protease, in primary breast cancer tissues has been correlated with increased risk of metastasis and shorter relapse-free survival in breast cancer patients. See, e.g., Gulnik et al., J. Mol. Biol., 227, 265–270 (1992). Increased levels of secretion of cathepsin D in breast cancer is due to both over overexpression of the gene and altered processing of the protein. High levels of cathepsin D and other proteases may degrade the extracellular matrix and thereby promote the escape of cancer cells to the lymphatic and circulatory system and enhance the invasion of new tissues. Most deaths incurred from cancer are due to its metastatic spread to secondary organs. It is therefore desirable to identify cathepsin D inhibitors as potential therapeutics for the prevention or treatment of cancer.
In view of the foregoing problems, there exists a need for new aspartic protease inhibitors, pharmaceutical compositions, and therapeutic methods of using aspartic protease inhibitors. The present invention provides such aspartic protease inhibitors, pharmaceutical compositions, and therapeutic methods of using them. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.